Algal channelrhodopsins are light-gated channels widely used for targeted photocontrol of neuron activity. Over the past six years, the temporal and spatial precision of their light-activation has proven to be incisive and powerful for research on brain circuitry, and more recently channelrhodopsin experimental therapy in animal models of neurological diseases has produced promising results. However, the extremely low ion conductance of channelrhodopsins limits their use and is a significant barrier for human clinical trials because it necessitates high level expression and high light intensities. Overexpression of a foreign membrane protein is detrimental to long-term cellular health and also creates an immune response risk. In stark contrast to the poor photosensitivity in neurons, channelrhodopsins in their native algal cells are low abundance photosensory receptors that mediate phototaxis in extremely low light with near single-photon sensitivity. This exquisite photosensitivity derives from the ~103-fold amplification of channelrhodopsin currents by secondary activation of highly conductive Ca2+ channels in the algal plasma membrane. We propose to develop a new class of ultrasensitive optogenetic tools with the ~103-fold greater light sensitivity by amplification of channelrhodopsin action by co-expression with the Ca2+ channels that naturally perform this function in the alga Chlamydomonas reinhardtii. Our approach is to identify molecular components responsible for amplification of channelrhodopsin-mediated photocurrents in the algae and to construct a combined channelrhodopsin-Ca2+ channel tool. We will apply genome-wide transcriptome profiling coupled to electrophysiological assay of the amplification process to identify the Ca2+ channels and other components necessary for amplification, if they exist. Our evidence strongly favors a direct interaction mechanism, so we expect to identify one or more Ca2+ channels capable of direct amplification by protein-protein interaction. We will further analyze their action by microRNA silencing, and use this information to re-create in mammalian cells an ultrasensitive channelrhodopsin-Ca2+ channel complex used by algae for dim-light phototaxis. We expect this new optogenetic tool to be able to be effective when expressed in the plasma membrane at protein levels well below those that stress human cells. These ultrasensitive molecular complexes will remove a significant barrier to their clinical use, as well as enhance research on neurocircuitry, enabling brain studies not possible with current technology. PUBLIC HEALTH RELEVANCE: Channelrhodopsins are photoactive proteins enabling light activation of neurons by combined optical and genetic techniques (optogenetics). Light-activation of channelrhodopsins has been used to map circuitry in mammalian brain tissue, as well as for experimental therapeutics, including restoration of vision in blind mice. This project seeks to develop new types of optogenetic tools with ~1000-fold higher sensitivity than the currently available ones, based on interaction of channelrhodopsins with specific Ca2+ channels. Such ultrasensitive light-activated channel complexes will enable biomedical research not possible with current technology and overcome a significant barrier to clinical use of optogenetics in humans.